Methods of hydrolyzing proteins are well known in the art. Conventionally, protein hydrolysates are produced chemically by hydrolyzing protein or proteinaceous material, such as for example defatted soy flour or wheat gluten, with hydrochloric acid under refluxing conditions. The resulting hydrolysates are inexpensive and can have satisfactory organoleptic properties. However, chemical hydrolysis is accompanied by non-specific side reactions causing e.g. the formation of chlorohydrins, such as monochlorodihydroxypropanols (MCDPs) and dichloropropanols (DCPs), the presence of which is undesirable in food products. The food industry is demanding milder methods of modifying food products, reducing the need for harsh, chemical reaction conditions and for removal of side-reaction products and residual reagents.
Alternatively, protein or proteinaceous material may be hydrolyzed enzymatically. Typically, the relevant protein source is first subjected to a (partial) hydrolysis with one or more suitable endoproteases. Then, the resulting protein fragments can be degraded completely or partially into individual amino acids or dipeptides or tripeptides by the use of exopeptidases. Alternatively, the endopeptidases and exopeptidases may function simultaneously in an enzyme mixture resulting in a similar complete or partial degradation of the protein or proteinaceous material.
A fundamental problem in the enzymatic hydrolysis of proteins and proteinaceous material is the formation of a bitter flavor due to the formation of short peptide fragments. The bitter taste is believed to be the result of cleavage of proteins at amino acids with hydrophobic side chains, resulting in the formation of peptides with exposed hydrophobic side chains, which are typically inaccessible in proteins and longer peptides due to their tertiary structure.
In order to solve this problem, the state of the art proposes to use specific proteases to limit the degree of hydrolysis and obtain preferred terminal side-chains. For example, U.S. Pat. No. 5,866,357 describes the use of a Glu/Asp specific protease for the preparation of hydrolysates, optionally with the use of an additional specific protease. Furthermore, WO 98/27827, for example, proposes, for the purpose of solving the problem, using a proteolytic enzyme mixture comprising only one exopeptidase, wherein the exopeptidase is produced using rDNA techniques, and may be employed in combination with one or more suitable endopeptidases, such as Fromase™ (Gist-Brocades, France) and Maxatase™ (Genencor International, Belgium). U.S. Pat. No. 5,532,007 discloses the use of a combination of purified enzymes, a neutral protease producible by means of a Bacillus strain and an alkaline protease producible by means of a Bacillus strain. The reference states that raw meat is preferably treated with proteases exclusively with endo-activities for the purpose of the described invention to obtain meat hydrolysates. Methods disclosed in WO 94/25580 employ a proteolytic enzyme preparation derived from the fungus Aspergillus oryzae (Flavorzyme™) comprising a mixture of endopeptidases and exopeptidases.
Alternatively, EP 0 823 998 A2, proposes the enzymatic hydrolysis of smoked meat as the protein or proteinaceous material for the generation of a protein hydrolysate which does not taste bitter. In this process it is preferred that only one enzyme, preferably a neutral or an alkaline protease having an endopeptidase effect, for example Pescalase (Gist Brocades), Alcalase (Novo Nordisk) or Promod 31 (Biocatalysts), be employed for the hydrolysis.
WO 89/10960 proposes the use of complex enzyme mixtures from krill to modificate protein, peptide and/or lipid constituents of biological material in industrial processes. The enzyme compositions disclosed are prepared by incubating macerated whole Antarctic krill (Euphasia superba) at 50° C. for 20 h. Examples of various applications are described, such as hydrolysis of fish and meat. The krill enzyme preparations are said to contain different proteases and lipolytic enzymes, e.g. a considerable amount of phospholipases. Such preparations do not seem to have been used on a commercial scale for the production of food-grade hydrolysates. It should be noted that in all of these prior art references, hydrolysates are obtained by enzymatic incubation at 50–65° C.
WPI abstract AN 1996495762 (RU-C-2 055 482) describes the use of pylloritic enzymes from Salmon for the production of protein-nucleic hydrolysates, but low-temperature hydrolysis is not discosed or anticipated. U.S. Pat. No. 3,852,479 describes a process for producing protein hydrolysates having a high glutamic acid content, by hydrolyzing protein material with a glutaminase together with a proteolytic enzyme. Also herein are temperature above 50° C. preferred. CA 131385 describes the use of porcine trypsin or Enzeco AP-1™ protease (alkaline phosphatase of bacterial origin) for enzymatic extraxtion of carotenoprotein from crustacean wastes. None of these references disclose the use of proteases from Gadiform fish species for producing protein hydrolysates or suggest that such or other similar enzyme compositions would be effective at low temperatures.
For the production of many delicate processed food products originating from or simulating natural products, such as, e.g. seafood soups and sauces, flavor extracts need not only have non-bitter properties but must also exhibit the same or a very similar flavor as the characterizing flavor of the natural product. Most quality-restaurants still prepare extracts for e.g. shellfish soups using conventional methods of making a stock from shells, claws, heads etc. Enzymatically prepared flavor agents have generally not been used widely as they do not seem to provide a satisfactory natural flavor.
The odors of fish and other seafood species are produced by complex mixtures of volatile compounds, and are very sensitive to conditions affecting the freshness of the species. Species-related odor compounds in fresh fish are present in very low levels, but many of such compounds have low odor thresholds and therefore, even though present in low levels (ppb) they still affect the overall aroma of the fish species and changes in their concentrations drastically affect the overall aroma. These compounds include unsaturated carbonyl compounds and alcohols with six, eight, or nine carbon atoms. Also, bromophenols in low concentrations have been associated with the natural, sea-, iodine, marine-like flavors of seafood. Microbial compounds are microbially formed during spoilage of fish. These include short chain alcohols, ketones, aldehydes, amines, sulphur compounds, aromatics and acids, and result mainly from the degradation of amino- and fatty acids. Proteolytic activity will accelerate the spoilage as small peptides and free amino acids are nutrients for bacteria, resulting in the formation of bad smelling metabolites. Therefore, careful selection of conditions for the hydrolysis of seafood and related protein materials is highly critical for the production of such hydrolysates for high-quality food products with desirable organoleptic properties. It is likely that prolonged incubations at temperatures in the range of 50–65° C. will degrade the overall flavor and aromatic character of the protein-containing materials being hydrolyzed, in particular of fish and other seafood material, due to the loss of volatile compounds and production of undesired side-reaction products.
There is a need for methods of hydrolyzing proteins under mild conditions leading to high yields and to hydrolysates with excellent organoleptic properties, in particular protein hydrolysates that have preserved the natural flavor of the protein-containing starting material such as seafood, but which do not have the bitter flavor which arises during conventional hydrolysis of protein-containing materials. It has been found that methods for obtaining hydrolysates at low enzyme incubation temperatures result in that the freshness and flavor (volatile flavor agents) of the raw material can be preserved, assumingly due to a reduced level of side-reactions and microbial activity and that a relatively non-specific enzyme preparation derived from fish intestines and obtainable with economical and technically simple methods can be used for effective hydrolysis of proteinaceous materials to obtain hydrolysates that are non-bitter and retain excellent flavor characteristics of the protein-containing raw material. A particularly advantageous aspect of the invention is the low temperature range at which such enzyme preparations are proteolytically active. It is conceivable that the low temperature range at which the methods and processes of the invention are preferably carried out at is an important factor contributing to the organoleptic properties of the obtained products.